Mixture-Dependent Toxicity of E-Cigarette Constituents: Cytotoxicity, Inflammation, and DNA-Repair-Associated Protein Responses in Bronchial Epithelial Cells

Electronic cigarette (e-cigarette) aerosols contain complex mixtures of flavoring chemicals, synthetic cooling agents, and metals derived from device components. The biological effects of these constituents in combination remain incompletely characterized. We evaluated selected flavoring chemicals and nickel oxide (NiO), individually and in combination, in BEAS-2B human bronchial epithelial cells. Cells were exposed for 24 h to vanillin, WS23, ethyl vanillin, or p-anisaldehyde over an experimental concentration range of 10–1000 µM. Vanillin-WS23 and ethyl vanillin-p-anisaldehyde mixtures were evaluated at a fixed total concentration of 1000 µM over multiple component ratios. Separate experiments evaluated 100 µM ethyl vanillin, 50 µg/cm2 NiO, and their combination. Vanillin, WS23, ethyl vanillin, and p-anisaldehyde reduced viability-associated metabolic activity and cellular ATP in a concentration-dependent manner. The lowest concentrations associated with significant reductions in the MTT endpoint were 200 µM for vanillin, 50 µM for WS23, 300 µM for ethyl vanillin, and 200 µM for p-anisaldehyde. Chou-Talalay analysis identified synergistic interactions between vanillin and WS23 (combination index [CI] < 1) and antagonistic interactions between ethyl vanillin and p-anisaldehyde (CI > 1). Ethyl vanillin and NiO produced no statistically significant decrease in viability but increased inflammatory cytokines in conditioned medium. Both IL-6 and IL-8 levels were significantly elevated with NiO and the combined ethyl vanillin-NiO exposure. Total H2AX abundance did not differ significantly among groups at 24 h. NEIL2 abundance showed increasing trends following NiO alone (p = 0.0518) and combined ethyl vanillin–NiO exposure (p = 0.0760). PNKP increased significantly after ethyl vanillin (p = 0.0261) and combined ethyl vanillin-NiO (p = 0.0041) treatments. These findings show mixture-dependent biological responses that differed from the effects observed with individual constituents. Flavoring chemical mixtures produced either synergistic or antagonistic effects, and ethyl vanillin-NiO exposure produced inflammatory and DNA-repair-associated protein responses under conditions that did not significantly reduce cell viability. By demonstrating non-additive flavoring chemical interactions together with inflammatory and DNA-repair-associated protein responses to chemical-metal exposure, this study evaluated e-cigarette toxicity beyond single-constituent hazard characterization. The NEIL2 and PNKP findings support altered base-excision repair (BER) pathways but do not establish oxidative DNA lesions or increased DNA repair activity.